In vitro evaluation of the carcinogenic potential of perfluorinated chemicals.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the major components of long-chain per- and polyfluorinated alkyl substances (PFAS), known for their chemical stability and environmental persistence. Even if PFOA and PFOS have been phased out or are limited in use, they still represent a concern for human and environmental health. Several studies have been performed to highlight the toxicological behavior of these chemicals and their mode of action (MoA). Data suggested the causal association between PFOA or PFOS exposure and carcinogenicity in humans, but the outcomes of epidemiological studies showed some inconsistency. Moreover, the hypothesized MoA based on animal studies is considered not relevant for human cancer. In order to improve the knowledge on PFAS toxicology and contribute to the weight of evidence for the regulatory classification of PFAS, we used the BALB/c 3T3 cell transformation assay (CTA), an in vitro model under consideration to be included in an integrated approach to testing and assessment for non-genotoxic carcinogens (NGTxCs). PFOS and PFOA were tested at several concentrations by using a validated experimental protocol. Our results demonstrated that PFOA is not able to induce cell transformation, whereas PFOS exposure led to a concentration-related increase of type-III foci. Malignant foci formation is triggered at PFOS concentrations equal to or higher than 50 ppm. It is not directly associated with cytotoxicity or proliferation induction. The divergent CTA outcomes suggest that different molecular events could be responsible for the toxicological profiles of PFOS and PFOA, which were not completely captured in our study.

This study explored PFOS and PFOA, common PFAS chemicals, to understand their potential harm and cancer risk. PFAS are known for their durability and resistance to heat, water and oil. They are persistent in the environment and may pose health risks despite decreased use. To better understand how PFOS and PFOA might be harmful, we conducted an in vitro test that can resemble the carcinogenesis process in experimental animals. Testing these chemicals on lab cells revealed PFOS can cause cancer-like changes, at levels of 50 parts per million or higher, but not PFOA. This difference suggests PFAS chemicals affect cells differently, but we need more research to understand exactly how they work and how they might cause cancer. Understanding this could help regulate and reduce PFAS harmful effects. Moreover, this research aligns with 3R principles by using cell-based tests as an alternative to animal testing, promoting ethical research practices.

Multipotent mesenchymal stromal cells derived from porcine exocrine pancreas improve insulin secretion from juvenile porcine islet cell clusters.

Neonatal and juvenile porcine islet cell clusters (ICC) present an unlimited source for islet xenotransplantation to treat type 1 diabetes patients. We isolated ICC from pancreata of 14 days old juvenile piglets and characterized their maturation by immunofluorescence and insulin secretion assays. Multipotent mesenchymal stromal cells derived from exocrine tissue of same pancreata (pMSC) were characterized for their differentiation potential and ability to sustain ICC insulin secretion in vitro and in vivo. Isolation of ICC resulted in 142 ± 50 × 103 IEQ per pancreas. Immunofluorescence staining revealed increasing presence of insulin-positive beta cells between day 9 and 21 in culture and insulin content per 500IEC of ICC increased progressively over time from 1178.4 ± 450 µg/L to 4479.7 ± 1954.2 µg/L from day 7 to 14, P < .001. Highest glucose-induced insulin secretion by ICC was obtained at day 7 of culture and reached a fold increase of 2.9 ± 0.4 compared to basal. Expansion of adherent cells from the pig exocrine tissue resulted in a homogenous CD90+ , CD34- , and CD45- fibroblast-like cell population and differentiation into adipocytes and chondrocytes demonstrated their multipotency. Insulin release from ICC was increased in the presence of pMSC and dependent on cell-cell contact (glucose-induced fold increase: ICC alone: 1.6 ± 0.2; ICC + pMSC + contact: 3.2 ± 0.5, P = .0057; ICC + pMSC no-contact: 1.9 ± 0.3; theophylline stimulation: alone: 5.4 ± 0.7; pMSC + contact: 8.4 ± 0.9, P = .013; pMSC no-contact: 5.2 ± 0.7). After transplantation of encapsulated ICC using Ca2+ -alginate (alg) microcapsules into streptozotocin-induced diabetic and immunocompetent mice, transient normalization of glycemia was obtained up to day 7 post-transplant, whereas ICC co-encapsulated with pMSC did not improve glycemia and showed increased pericapsular fibrosis. We conclude that pMSC derived from juvenile porcine exocrine pancreas improves insulin secretion of ICC by direct cell-cell contact. For transplantation purposes, the use of pMSC to support beta-cell function will depend on the development of new anti-fibrotic polymers and/or on genetically modified pigs with lower immunogenicity.

Prolongation of rat-to-mouse islets xenograft survival by co-transplantation of autologous IL-10 differentiated murine tolerogenic dendritic cells.

BACKGROUND: Tolerogenic dendritic cells (DCs) represent a promising approach to promote transplantation tolerance. In this study, the potential of autologous bone marrow (BM)-derived murine DC to protect rat-to-mouse islets xenografts was analyzed. METHODS: Tolerogenic DCs were generated by differentiating BM cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin 10 (IL-10, IL-10 DC). The phenotype of IL-10 DC was characterized in vitro by expression of costimulatory/inhibitory molecules (flow cytometry) and cytokines (Luminex and ELISA), their function by phagocytosis and T-cell stimulation assays. To study transplant tolerance in vivo, rat islets were transplanted alone or in combination with autologous murine IL-10 DC under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. Xenograft survival was evaluated by monitoring glycemia, cellular infiltration of xenografts by microscopy and flow cytometry 10 days post-transplantation. RESULTS: Compared with control DC, IL-10 DC exhibited lower levels of major histocompatibility complex class II, costimulatory molecules (CD40, CD86, CD205), lower production of pro-inflammatory cytokines (IL-12p70, TNF, IL-6), and higher production of IL-10. Phagocytosis of xenogeneic rat splenocytes was not impaired in IL-10 DC, whereas stimulation of T-cell proliferation was reduced in the presence of IL-10 DC. Xenograft survival of rat islets in diabetic mice co-transplanted with autologous murine IL-10 DC was significantly prolonged from 12 to 21 days, without additional immunosuppressive treatment. Overall, infiltration of xenografts by T cells and myeloid cells was not different in IL-10 DC recipient mice, but enriched for CD8+ T cells and myeloid cells with suppressor-associated phenotype. CONCLUSIONS: Autologous IL-10-differentiated DC with tolerogenic properties prolong rat-to-mouse islets xenograft survival, potentially by locally inducing immune regulatory cells, indicating their potential for regulatory immune cell therapy in xenotransplantation.

Immunological aspects of allogeneic pancreatic islet transplantation: a comparison between mouse and human.

Pancreatic islet allotransplantation is a treatment for patients with severe forms of type 1 diabetes. As long-term graft function and survival are not yet optimal, additional studies are warranted in order to continue improving transplant outcomes. The mechanisms of islet graft loss and tolerance induction are often studied in murine diabetes models. Despite numerous islet transplantation studies successfully performed over recent years, translation from experimental mouse models to human clinical application remains elusive. This review aims at critically discussing the strengths and limitations of current mouse models of diabetes and experimental islet transplantation. In particular, we will analyze the causes leading to diabetes and compare the immunological mechanisms responsible for rejection between mouse and human. A better understanding of the experimental mouse models should facilitate translation to human clinical application.

Interleukin-1 Receptor Antagonist Modulates Liver Inflammation and Fibrosis in Mice in a Model-Dependent Manner.

BACKGROUND: Interleukin-1 (IL-1)ß and IL-1 receptor antagonist (IL-1Ra) have been proposed as important mediators during chronic liver diseases. We aimed to determine whether the modulation of IL-1ß signaling with IL-1Ra impacts on liver fibrosis. METHODS: We assessed the effects of IL-1ß on human hepatic stellate cells (HSC) and in mouse models of liver fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride treatment (CCl-4). RESULTS: Human HSCs treated with IL-1ß had increased IL-1ß, IL-1Ra, and MMP-9 expressions in vitro. HSCs treated with IL-1ß had reduced α-smooth muscle actin expression. These effects were all prevented by IL-1Ra treatment. In the BDL model, liver fibrosis and Kuppfer cell numbers were increased in IL-1Ra KO mice compared to wild type mice and wild type mice treated with IL-1Ra. In contrast, after CCl-4 treatment, fibrosis, HSC and Kupffer cell numbers were decreased in IL-1Ra KO mice compared to the other groups. IL-1Ra treatment provided a modest protective effect in the BDL model and was pro-fibrotic in the CCl-4 model. CONCLUSIONS: We demonstrated bivalent effects of IL-1Ra during liver fibrosis in mice. IL-1Ra was detrimental in the CCl-4 model, whereas it was protective in the BDL model. Altogether these data suggest that blocking IL-1-mediated inflammation may be beneficial only in selective liver fibrotic disease.

Antifibrotic Effect of Ketoprofen-Grafted Alginate Microcapsules in the Transplantation of Insulin Producing Cells.

The controlled release of small molecular modulators of the immune response from hydrogel microspheres (MS) used for cell immobilization is an attractive approach to reduce pericapsular fibrotic overgrowth (PFO) after transplantation. Ketoprofen is a well-known nonsteroidal anti-inflammatory drug involved in the early stage inflammation cascade. PEGylated derivatives of ketoprofen, presenting either ester or amide linkage to the drug, were synthesized and conjugated to the hydroxyl groups of sodium alginate (Na-alg). Functionalized cell-free and MIN6 cells containing MS were produced from the resulting modified alginates. In vitro quantification of ketoprofen release indicated regular and sustained drug delivery over 14 days, resulting from the hydrolytic cleavage of the ester bond. The release kinetics was enhanced over the initial 7 days by the presence of MIN6 cells, probably as a result of cell esterase activity. In the presence of amide bond, traces of ketoprofen were released over 14 days due to a much slower hydrolysis kinetics. Cell-free and MIN6 cells containing MS were transplanted in immune-competent mice, either in the peritoneal cavity or under the kidney capsule, with a follow-up period of 30 days. Comparison with nonmodified Ca-alg MS transplanted in the same conditions demonstrated a clear reduction in the severity of PFO for MS functionalized with ketoprofen. Quantification of collagen deposition on MIN6 cells containing MS transplanted under the kidney capsule revealed the significant effect of ketoprofen release to decrease fibrotic tissue formation. The impact was more pronounced when the drug was covalently conjugated by an ester linkage, allowing higher concentration of the anti-inflammatory compound to be delivered at the transplantation site. The functionality of microencapsulated MIN6 cells 30 days after transplantation was confirmed by detection of insulin positive cell content.

Beneficial Effects of Human Mesenchymal Stromal Cells on Porcine Hepatocyte Viability and Albumin Secretion.

Porcine hepatocytes transplanted during acute liver failure might support metabolic functions until the diseased liver recovers its function. Here, we isolated high numbers of viable pig hepatocytes and evaluated hepatocyte functionality after encapsulation. We further investigated whether coculture and coencapsulation of hepatocytes with human multipotent mesenchymal stromal cells (MSC) are beneficial on hepatocyte function. Livers from 10 kg pigs (n = 9) were harvested, and hepatocytes were isolated from liver suspensions for microencapsulation using alginate and poly(ethylene-glycol)- (PEG-) grafted alginate hydrogels, either alone or in combination with MSC. Viability, albumin secretion, and diazepam catabolism of hepatocytes were measured for one week. 9.2 ± 3.6 × 109 hepatocytes with 95.2 ± 3.1% viability were obtained after isolation. At day 3, free hepatocytes displayed 99% viability, whereas microencapsulation in alginate and PEG-grafted alginate decreased viability to 62% and 48%, respectively. Albumin secretion and diazepam catabolism occurred in free and microencapsulated hepatocytes. Coencapsulation of hepatocytes with MSC significantly improved viability and albumin secretion at days 4 and 8 (p < 0.05). Coculture with MSC significantly increased and prolonged albumin secretion. In conclusion, we established a protocol for isolation and microencapsulation of high numbers of viable pig hepatocytes and demonstrated that the presence of MSC is beneficial for the viability and function of porcine hepatocytes.

Multipotent mesenchymal stromal cells enhance insulin secretion from human islets via N-cadherin interaction and prolong function of transplanted encapsulated islets in mice.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) enhance viability and function of islets of Langerhans. We aimed to examine the interactions between human MSC and human islets of Langerhans that influence the function of islets. METHODS: Human MSC and human islets (or pseudoislets, obtained after digestion and reaggregation of islet cells) were cocultured with or without cellular contact and glucose-stimulated insulin secretion assays were performed to assess cell function. The expression of several adhesion molecules, notably ICAM-1 and N-cadherin on islets and MSC, was investigated by qPCR. The role of N-cadherin was analyzed by adding an anti-N-cadherin antibody in islets cultured with or without MSC for 24 h followed by insulin measurements in static incubation assays. Islets and MSC were coencapsulated in new hydrogel microspheres composed of calcium alginate and covalently crosslinked polyethylene glycol. Encapsulated cells were transplanted intraperitoneally in streptozotocin-induced diabetic mice and glycemia was monitored. Islet function was evaluated by the intraperitoneal glucose tolerance test. RESULTS: In vitro, free islets and pseudoislets cocultured in contact with MSC showed a significantly increased insulin secretion when compared to islets or pseudoislets cultured alone or cocultured without cell-to-cell contact with MSC (p < 0.05). The expression of ICAM-1 and N-cadherin was present on islets and MSC. Blocking N-cadherin prevented the enhanced insulin secretion by islets cultured in contact with MSC whereas it did not affect insulin secretion by islets cultured alone. Upon transplantation in diabetic mice, islets microencapsulated together with MSC showed significantly prolonged normoglycemia when compared with islets alone (median 69 and 39 days, respectively, p < 0.01). The intraperitoneal glucose tolerance test revealed an improved glycemic response in mice treated with islets microencapsulated together with MSC compared to mice transplanted with islets alone (p < 0.001). CONCLUSIONS: MSC improve survival and function of islets of Langerhans by cell-to-cell contact mediated by the adhesion molecule N-cadherin.

Synthesis Strategies to Extend the Variety of Alginate-Based Hybrid Hydrogels for Cell Microencapsulation.

The production of hydrogel microspheres (MS) for cell immobilization, maintaining the favorable properties of alginate gels but presenting enhanced performance in terms of in vivo durability and physical properties, is desirable to extend the therapeutic potential of cell transplantation. A novel type of hydrogel MS was produced by straightforward functionalization of sodium alginate (Na-alg) with heterotelechelic poly(ethylene glycol) (PEG) derivatives equipped with either end thiol or 1,2-dithiolane moieties. Activation of the hydroxyl moieties of the alginate backbone in the form of imidazolide intermediate allowed for fast conjugation to PEG oligomers through a covalent carbamate linkage. Evaluation of the modified alginates for the preparation of MS combining fast ionic gelation ability of the alginate carboxylate groups and slow covalent cross-linking provided by the PEG-end functionalities highlighted the influence of the chemical composition of the PEG-grafting units on the physical characteristics of the MS. The mechanical properties of the MS (resistance and shape recovery) and durability of PEG-grafted alginates in physiological environment can be adjusted by varying the nature of the end functionalities and the length of the PEG chains. In vitro cell microencapsulation studies and preliminary in vivo assessment suggested the potential of these hydrogels for cell transplantation applications.

Microencapsulation of Hepatocytes and Mesenchymal Stem Cells for Therapeutic Applications.

Encapsulated hepatocyte transplantation and encapsulated mesenchymal stem cell transplantation are newly developed potential treatments for acute and chronic liver diseases, respectively. Cells are microencapsulated in biocompatible semipermeable alginate-based hydrogels. Microspheres protect cells against antibodies and immune cells, while allowing nutrients, small/medium size proteins and drugs to diffuse inside and outside the polymer matrix. Microencapsulated cells are assessed in vitro and designed for experimental transplantation and for future clinical applications.Here, we describe the protocol for microencapsulation of hepatocytes and mesenchymal stem cells within hybrid poly(ethylene glycol)-alginate hydrogels.

Cell rearrangement in transplanted human islets.

The major feature of the human pancreatic islet architecture is the organization of endocrine cells into clusters comprising central ß cells and peripheral α cells surrounded by vasculature. To have an insight into the mechanisms that govern this unique islet architecture, islet cells were isolated, and reaggregation of α and ß cells into islet-like structures (pseudoislets) after culture or transplantation into mice was studied by immunohistology. The pseudoislets formed in culture displayed an unusual cell arrangement, contrasting with the transplanted pseudoislets, which exhibited a cell arrangement similar to that observed in native pancreatic islet subunits. The pattern of revascularization and the distribution of extracellular matrix around transplanted pseudoislets were alike to those observed in native pancreatic islets. This organization of transplanted pseudoislets occurred also when revascularization was abolished by treating mice with an anti-VEGF antibody, but not when contact with extracellular matrix was prevented by encapsulation of pseudoislets within alginate hydrogel. These results indicate that the maintenance of islet cell arrangement is dependent on in vivo features such as extracellular matrix but independent of vascularization.

IL-22 capacitates dermal fibroblast responses to TNF in scleroderma.

OBJECTIVES: Interleukin (IL) 22 mRNA in systemic sclerosis (SSc) skin and Th22 cells in SSc peripheral blood are increased, but the role of IL-22 in fibrosis development remains poorly understood. METHODS: Biopsies were obtained from the involved skin of 15 SSc, 4 morphea and 8 healthy donors (HD). The presence of IL-22+ cells in the skin was determined by immunostaining. The in vitro response of HD and SSc fibroblasts to IL-22, IL-22 in conjunction with tumour necrosis factor (TNF) or keratinocyte conditioned medium was assessed by ELISA, radioimmunoassay (RIA), real-time PCR and western blot. The in vivo response in mice was assessed by histomorphometry. RESULTS: IL-22+ cells were over-represented in the dermis and epidermis of morphea and in the epidermis of SSc compared with HD. The majority of dermal IL-22+ cells were T cells. Dermal fibroblasts expressed both IL-22 receptor subunits IL-10RB and IL-22RA, expression of which was enhanced by TNF and reduced by transforming growth factor (TGF)-ß. IL-22 induced rapid phosphorylation of p38 and ERK1/2 in fibroblasts, but failed to induce the synthesis of chemokines and extracellular matrix components. However, IL-22 enhanced the production of monocyte chemotactic protein 1, IL-8 and matrix metalloproteinase 1 induced by TNF. Fibroblast responses were maximal in the presence of conditioned medium from keratinocytes activated by IL-22 in conjunction with TNF. Dermal thickness was maximal in mice injected simultaneously with IL-22 and TNF. CONCLUSIONS: IL-22 capacitates fibroblast responses to TNF and promotes a proinflammatory fibroblast phenotype by favouring TNF-induced keratinocyte activation. These results define a novel role for keratinocyte-fibroblast interactions in the context of skin fibrosis.

Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice.

BACKGROUND & AIMS: Mesenchymal stem cell (MSC) transplantation was shown to be effective for the treatment of liver fibrosis, but the mechanisms of action are not yet fully understood. We transplanted encapsulated human MSCs in two mouse models of liver fibrosis to determine the mechanisms behind the protective effect. METHODS: Human bone marrow-derived MSCs were microencapsulated in novel alginate-polyethylene glycol microspheres. In vitro, we analyzed the effect of MSC-conditioned medium on the activation of hepatic stellate cells and the viability, proliferation, cytokine secretion, and differentiation capacity of encapsulated MSCs. The level of fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride (CCl4) was assessed after intraperitoneal transplantation of encapsulated MSCs, encapsulated human fibroblasts, and empty microspheres. RESULTS: MSC-conditioned medium inhibited hepatic stellate cell activation and release of MSC secreted anti-apoptotic (IL-6, IGFBP-2) and anti-inflammatory (IL-1Ra) cytokines. Viability, proliferation, and cytokine secretion of microencapsulated MSCs were similar to those of non-encapsulated MSCs. Within the microspheres, MSCs maintained their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. 23% (5/22) of the MSC clones were able to produce anti-inflammatory IL-1Ra in vitro. Microencapsulated MSCs significantly delayed the development of BDL- and CCl4-induced liver fibrosis. Fibroblasts had an intermediate effect against CCl4-induced fibrosis. Mice transplanted with encapsulated MSCs showed lower mRNA levels of collagen type I, whereas levels of matrix metalloproteinase 9 were significantly higher. Human IL-1Ra was detected in the serum of 36% (4/11) of the mice transplanted with microencapsulated MSCs. CONCLUSIONS: MSC-derived soluble molecules are responsible for an anti-fibrotic effect in experimental liver fibrosis.

High IL-17E and low IL-17C dermal expression identifies a fibrosis-specific motif common to morphea and systemic sclerosis.

BACKGROUND: High interleukin (IL)-17A levels are characteristically found in the skin of systemic sclerosis (SSc) individuals. Our aim was to investigate whether the dermal expression of IL-17A and related IL-17 family members (i.e. IL-17C, IL-17E and IL-17F) could distinguish fibrotic from healthy skin and could show similarities in SSc and morphea, two disorders with presumed distinct pathogenesis, but characterized by skin fibrosis. METHODS: Biopsies were obtained from the involved skin of 14 SSc, 5 morphea and 8 healthy donors (HD) undergoing plastic surgery. Immunohistochemistry/immunofluorescence techniques were coupled to a semi-automated imaging quantification approach to determine the presence of the IL-17 family members in the skin. The in vitro effects induced by the IL-17 family members on fibroblasts from normal and SSc individuals were assessed by ELISA and RIA. RESULTS: Positive cells for each of the IL-17 isoforms investigated were present in the dermis of all the individuals tested, though with variable frequencies. SSc individuals had increased frequency of IL-17A+ (p = 0.0237) and decreased frequency of IL-17F+ (p = 0.0127) and IL-17C+ cells (p = 0.0008) when compared to HD. Similarly, morphea individuals had less frequent IL-17C+ cells (p = 0.0186) in their skin but showed similar number of IL-17A+ and IL-17F+ cells when compared to HD. Finally, IL-17E+ cells were more numerous in morphea (p = 0.0109) and tended to be more frequent in SSc than in HD. Fibroblast production of IL-6, MMP-1 and MCP-1 was enhanced in a dose-dependent manner in the presence of IL-17E and IL-17F, but not in the presence of IL-17C. None of the cytokine tested had significant effect on type I collagen production. Of interest, in SSc the frequency of both IL-17A and IL-17F positive cells increased with disease duration. CONCLUSIONS: The frequency of IL-17A and IL-17F distinguish SSc to morphea individuals while dermal expression of IL-17C (low) and IL-17E (high) identifies a fibrosis-specific motif. The specific IL-17C/IL-17E cytokine combination may thus play a role in the development of fibrosis.

Survival of free and encapsulated human and rat islet xenografts transplanted into the mouse bone marrow.

Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.

Th17 cells favor inflammatory responses while inhibiting type I collagen deposition by dermal fibroblasts: differential effects in healthy and systemic sclerosis fibroblasts.

INTRODUCTION: T helper (Th)-17 cells are increased in systemic sclerosis (SSc). We therefore assessed whether Th17 cells could modulate the inflammatory and fibrotic responses in dermal fibroblasts from healthy donors (HD) and SSc individuals. METHODS: Fibroblasts were obtained from 14 SSc and 8 HD skin biopsies. Th17 clones were generated from healthy peripheral blood upon enrichment of CC chemokine receptor (CCR)-4/CCR6/CD161 expressing cells. Their cytokine production was assessed by flow cytometry and multiplex beads immunoassay. Fibroblast production of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8, matrix metalloproteinase (MMP)-1, tissue inhibitor of metalloproteinase (TIMP)-1, MMP-2 and type-I collagen was quantified by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), and changes in their transcription levels assessed by real-time PCR. Intracellular signals were dissected by western blot and the use of pharmacological inhibitors. IL-17A, tumor necrosis factor (TNF) and interferon-gamma (IFN-γ) blocking reagents were used to assess the specificity of the observed effects. RESULTS: IL-17A increased MCP-1, IL-8 and MMP-1 production in a dose-dependent manner while having no effect on type I collagen in HD and SSc fibroblasts both at protein and mRNA levels. Nuclear factor-kappa B (NF-κB) and p38 were preferentially involved in the induction of MCP-1 and IL-8, while MMP-1 was most dependent on c-Jun N-terminal kinase (JNK). Supernatants of activated Th17 clones largely enhanced MCP-1, IL-8 and MMP-1 while strongly inhibiting collagen production. Of note, the production of MCP-1 and IL-8 was higher, while collagen inhibition was lower in SSc compared to HD fibroblasts. The Th17 clone supernatant effects were mostly dependent on additive/synergistic activities between IL-17A, TNF and in part IFN-γ. Importantly, the inhibition of type I collagen production induced by the Th17 clone supernatants was completely abrogated by blockade of IL-17A, TNF and IFN-γ mostly in SSc fibroblasts, revealing an intrinsic resistance to inhibitory signals in SSc. CONCLUSIONS: Our findings demonstrate that in vitro Th17 cells elicit pro-inflammatory responses while restraining collagen production. Thus, the increased Th17 cell number observed in SSc may impact on the inflammatory component of the disease simultaneously potentially providing a protective role against fibrosis.

Interleukin-17A+ cell counts are increased in systemic sclerosis skin and their number is inversely correlated with the extent of skin involvement.

OBJECTIVE: Levels of interleukin-17A (IL-17A) have been found to be increased in synovial fluid from individuals with systemic sclerosis (SSc). This study was undertaken to investigate whether IL-17A-producing cells are present in affected SSc skin, and whether IL-17A exerts a role in the transdifferentiation of myofibroblasts. METHODS: Skin biopsy samples were obtained from the involved skin of 8 SSc patients and from 8 healthy control donors undergoing plastic surgery. Immunohistochemistry and multicolor immunofluorescence techniques were used to identify and quantify the cell subsets in vivo, including IL-17A+, IL-4+, CD3+, tryptase-positive, α-smooth muscle actin (α-SMA)-positive, myeloperoxidase-positive, and CD1a+ cells. Dermal fibroblast cell lines were generated from all skin biopsy samples, and quantitative polymerase chain reaction, Western blotting, and solid-phase assays were used to quantify α-SMA, type I collagen, and matrix metalloproteinase 1 (MMP-1) production by the cultured fibroblasts. RESULTS: IL-17A+ cells were significantly more numerous in SSc skin than in healthy control skin (P = 0.0019) and were observed to be present in both the superficial and deep dermis. Involvement of both T cells and tryptase-positive mast cells in the production of IL-17A was observed. Fibroblasts positive for α-SMA were found adjacent to IL-17A+ cells, but not IL-4+ cells. However, IL-17A did not induce α-SMA expression in cultured fibroblasts. In the presence of IL-17A, the α-SMA expression induced in response to transforming growth factor ß was decreased, while MMP-1 production was directly enhanced. Furthermore, the frequency of IL-17A+ cells was higher in the skin of SSc patients with greater severity of skin fibrosis (lower global skin thickness score). CONCLUSION: IL-17A+ cells belonging to the innate and adaptive immune system are numerous in SSc skin. IL-17A participates in inflammation while exerting an inhibitory activity on myofibroblast transdifferentiation. These findings are consistent with the notion that IL-17A has a direct negative-regulatory role in the development of dermal fibrosis in humans.

Prostaglandin I(2) analogues enhance already exuberant Th17 cell responses in systemic sclerosis.

OBJECTIVE: Among pleiotropic effects, the capacity of prostaglandin I(2) (PGI(2)) analogues to affect adaptive immunity remains poorly characterised. The purpose of this study was to assess whether PGI(2) analogues could affect T helper (Th) cell responses in patients with systemic sclerosis (SSc) and healthy donors (HD). METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from 33 patients with SSc and 29 HD. Cytokine levels in PBMC and monocyte/CD4 T cell cultures were quantified by immunoassays. The frequencies of interleukin (IL)-17A, IL-22, interferon γ (IFNγ) and IL-4-producing CD4 T cells were assessed by multiparametric flow cytometry. Selective receptor antagonists, cytokine blocking antibodies and signalling protein inhibitors were used to identify the receptors and signalling pathways mediating PGI(2) analogue effects. RESULTS: Th17 and Th22 cells were more abundant in individuals with SSc than in HD. PGI(2) analogues (iloprost, treprostinil and beraprost) significantly increased IL-17A and IL-22 in vitro while decreasing IFNγ production both in SSc and HD PBMC. These effects relied on the specific expansion of Th17 and Th22 and inhibition of Th1 cells. The enhanced Th17 cell responses depended on increased IL-23 production by monocytes, involved the IP prostacyclin receptor and required protein kinase A activation. Importantly, in vivo administration of iloprost in individuals with SSc presenting with digital ulcers resulted in a significant increase in the frequency of Th17 cells. CONCLUSIONS: These findings demonstrate that PGI(2) analogues affect Th cell differentiation/expansion programmes, favouring Th17 and inhibiting Th1 cell responses in SSc. The impact of these changes on the disease course needs to be taken into consideration and further exploited to improve SSc.

Increased frequency of circulating Th22 in addition to Th17 and Th2 lymphocytes in systemic sclerosis: association with interstitial lung disease.

INTRODUCTION: T cell abnormalities have been associated with the pathogenesis of systemic sclerosis (SSc). Recently, besides T helper (Th)17 cells, the Th22 subset has been identified in humans. Our purpose was to investigate the pattern of cytokines produced and chemokine-receptors expressed by peripheral blood (PB) Th cells in SSc and healthy donors (HD) focusing on cells producing interleukin (IL)-17 and IL-22 and to identify specific clinical associations. METHODS: Clinical data and peripheral blood were collected in 33 SSc individuals and 29 HD. IL-17A, IL-22, interferon gamma (IFN-γ), IL-4 production, the chemokine receptors CCR4, CCR6, CCR10, CXCR3 expression and the CD161 Th17 cell marker were assessed by multiparametric flow cytometry in PB CD4+ T cells. Intracellular cytokine accumulation was further investigated in CD4+ T cells expanded in vitro for seven days. RESULTS: The frequency of Th22, Th17, Th2, but not Th1 cells, was significantly increased in SSc individuals compared to HD. The percentage of CD161+CD4+ T cells was increased in SSc and correlated with the percentage of IL-17A producing cells. Moreover, the expression of the skin- and lung-homing chemokine receptor CCR6 correlated with the frequency of IL-22 and IL-17A-producing cells in SSc but not in HD. Finally, SSc interstitial lung disease (ILD) was strongly associated with higher numbers of IL-22 and, to a lesser extent, IL-17A-producing cells. CONCLUSIONS: IL-22 and IL-17A-producing T cells with skin- and lung-homing capabilities are characteristically increased in SSc. These findings support the hypothesis that Th22, in addition to Th17 cells, may be involved in pathological processes leading to SSc. While the association between IL-22 producing cells and ILD needs to be assessed in larger cohorts of patients, the increased frequency of Th22 cells appears to be a useful novel biomarker in SSc.

Fibrosis and immune dysregulation in systemic sclerosis.

Autoimmune and inflammatory phenomena are characteristically present in systemic sclerosis (SSc) and impact on dysregulated fibroblast extracellular matrix deposition, hallmark of the disease in conjunction with fibroproliferative vasculopathy. Oligoclonal T helper 2-like cells are present in the skin and peripheral blood in early diffuse disease. Type 2 cytokines synergize with profibrotic cytokines including transforming growth factor beta, favoring collagen deposition and metalloproteinase inhibition by fibroblasts. Furthermore, chemokine with pro-fibrotic and pro-angiogenic properties are preferentially produced by fibroblasts under the influence of Th2-like cells. The profibrotic monocyte chemotactic protein 1 is also produced by fibroblasts, partially in response to Toll-like receptor 4 (TLR4) recognition, when autoantibodies (autoAb) bind to fibroblast surface. In addition, immune-complex formed by autoAb and ubiquitous antigens including topoisomerase-1 favor the production of interferon-alpha (IFN-α) possibly by interacting with intravesicular TLRs. Consistent with this findings, unbiased gene screening has revealed that SSc peripheral blood cells express genes induced by IFN-α, a characteristic shared with systemic lupus erythematosus and other autoimmune disorders. These findings highlight the complex relationship between adaptive and acquired immune responses, which may participate to the pathogenesis of SSc in manners until now unsuspected, which may help in identifying novel therapeutic targets.